EN ISO 13885-3:2021
(Main)Gel permeation chromatography (GPC) - Part 3: Water as eluent (ISO 13885-3:2020)
Gel permeation chromatography (GPC) - Part 3: Water as eluent (ISO 13885-3:2020)
This document specifies the determination of the molar-mass distribution and the average molar mass values Mn (number average) and Mw (weight average) of polymers that are soluble in water by gel permeation chromatography (GPC).
NOTE Also known as size exclusion chromatography (SEC).
This method is applicable to neutral polymers and polyanions (e.g. polycarboxylates, polysaccharides, fully hydrolyzed polyvinyl alcohols and high-molecular polyethylene oxides). It is not applicable to polycations [e.g. polyvinylpyrrolidone, polyvinylpyridine, salts of poly(diallyl‑N,N‑dimethyl‑azacyclopentane), chitosan].
Despite good solubility in the mobile phase and even though the chromatograms obtained show good repeatability, it is possible that this method cannot be used with certain polymer types because of specific interactions (e.g. adsorption) within the sample/eluent/column system (see also Clause 12).
The conditions specified in this document are not applicable to the GPC analysis of polymer samples with Mw values greater than 106 g/mol and/or polymers with elution limits outside the calibration range (see 7.6 and Annex C).
This document includes no correction methods (e.g. for the elimination of peak broadening). If absolute molar mass values are required, an absolute method (e.g. membrane osmometry for Mn or light scattering for Mw) can be used.
Gelpermeationschromatographie (GPC) - Teil 3: Wasser als Elutionsmittel (ISO 13885-3:2020)
Dieses Dokument legt die Ermittlung der Molmassenverteilung und der Molmassenmittelwerte Mn (Zahlenmittel) und Mw (Gewichtsmittel) von wasserlöslichen Polymeren durch Gelpermeationschromatographie (GPC) fest.
ANMERKUNG Im Englischen auch als „size exclusion chromatography“ (SEC), übersetzt „Größenausschlusschromatographie“, bekannt.
Dieses Verfahren ist anwendbar auf neutrale Polymere und Polyanionen (z. B. Polycarboxylate, Polysaccharide, vollverseifte Polyvinylalkohole und hochmolekulare Polyethylenoxide). Es ist nicht anwendbar auf Polykationen [z. B. Polyvinylpyrrolidon, Polyvinylpyridin, Salze des Poly(diallyl-N,N-dimethyl-azacyclopentan), Chitosan].
Trotz guter Löslichkeit in der mobilen Phase und guter Wiederholbarkeit der resultierenden Chromatogramme ist es auf Grund spezifischer Wechselwirkungen (z. B. Adsorption) in dem System aus Probe/Elutionsmittel/Trennmaterial möglich, dass dieses Verfahren auf einzelne Polymere nicht angewandt werden kann (siehe auch Abschnitt 12).
Die in diesem Dokument festgelegten Bedingungen sind nicht anwendbar bei GPC-Analysen von Polymerproben mit Mw-Werten größer 106 g/mol und/oder von Polymeren, deren Elutionsgrenzen außerhalb des Kalibrierbereiches liegen (siehe 7.6 und Anhang C).
Dieses Dokument umfasst keine Korrekturverfahren (z. B. Elimination der Peakverbreiterung). Wenn absolute Molmassenwerte benötigt werden, kann auf ein Absolutverfahren (z. B. Membranosmometrie für Mn oder Lichtstreuung für Mw) zurückgegriffen werden.
WARNUNG - Dieses Dokument kann den Umgang mit gefährlichen Substanzen und Ausrüstungsteilen und die Ausführung gefährlicher Arbeitsgänge einschließen. Es erhebt nicht den Anspruch, auf alle gegebenenfalls vorhandenen und mit seiner Anwendung verbundenen Sicherheitsprobleme hinzuweisen. Es liegt in der Verantwortung des Anwenders, geeignete Vorkehrungen für den Arbeits‑ und Gesundheitsschutz zu treffen und die Übereinstimmung mit jeglichen nationalen Bestimmungen sicherzustellen.
Chromatographie par perméation de gel (GPC) - Partie 3: Eluant à l'eau (ISO 13885-3:2020)
Le présent document spécifie la détermination de la distribution de masses molaires et des valeurs de masse molaire moyenne Mn (moyenne en nombre) et Mw (moyenne en masse) de polymères solubles dans l’eau par chromatographie de perméation de gel (GPC, de l’anglais «gel permeation chromatography »).
NOTE Également appelée chromatographie d’exclusion stérique (SEC, de l’anglais «size exclusion chromatography »).
Cette méthode est applicable aux polymères neutres et aux polyanions [par exemple, polycarboxylates, polysaccharides, alcools polyvinyliques totalement hydrolysés et poly(oxyde d’éthylène) à masse moléculaire élevée]. Elle n’est pas applicable aux polycations [par exemple, polyvinylpyrrolidone, polyvinylpyridine, sels de poly(diallyl-N,N-diméthyl-azacyclopentane), chitosane].
Malgré une bonne solubilité dans la phase mobile et même si les chromatogrammes obtenus présentent une bonne répétabilité, il est possible que cette méthode ne puisse pas être utilisée avec certains types de polymère du fait d’interactions spécifiques (par exemple, adsorption) dans le système échantillon/éluant/colonne (voir également Article 12).
Les conditions spécifiées dans le présent document ne sont pas applicables à l’analyse GPC d’échantillons de polymères présentant des valeurs Mw supérieures à 106 g/mol et/ou de polymères dont les limites d’élution se situent en dehors de la gamme d’étalonnage (voir 7.6 et Annexe C).
Le présent document n’inclut pas de méthode de correction (par exemple, pour l’élimination d’un élargissement des pics). Si des valeurs absolues de masse molaire sont exigées, une méthode absolue (par exemple, osmométrie à membrane pour Mn ou diffusion de lumière pour Mw) peut être utilisée.
Gelska permeacijska kromatografija (GPC) - 3. del: Voda kot izpiralna tekočina (eluent) (ISO 13885-3:2020)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-november-2021
Gelska permeacijska kromatografija (GPC) - 3. del: Voda kot izpiralna tekočina
(eluent) (ISO 13885-3:2020)
Gel permeation chromatography (GPC) - Part 3: Water as eluent (ISO 13885-3:2020)
Gelpermeationschromatographie (GPC) - Teil 3: Wasser als Elutionsmittel (ISO 13885-
3:2020)
Chromatographie par perméation de gel (GPC) - Partie 3: Eluant à l'eau (ISO 13885-
3:2020)
Ta slovenski standard je istoveten z: EN ISO 13885-3:2021
ICS:
87.060.20 Veziva Binders
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 13885-3
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2021
EUROPÄISCHE NORM
ICS 87.060.20
English Version
Gel permeation chromatography (GPC) - Part 3: Water as
eluent (ISO 13885-3:2020)
Chromatographie par perméation de gel (GPC) - Partie Gelpermeationschromatographie (GPC) - Teil 3:
3: Eluant à l'eau (ISO 13885-3:2020) Wasser als Elutionsmittel (ISO 13885-3:2020)
This European Standard was approved by CEN on 23 August 2021.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 13885-3:2021 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
The text of ISO 13885-3:2020 has been prepared by Technical Committee ISO/TC 35 "Paints and
varnishes” of the International Organization for Standardization (ISO) and has been taken over as
which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by March 2022, and conflicting national standards shall
be withdrawn at the latest by March 2022.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 13885-3:2020 has been approved by CEN as EN ISO 13885-3:2021 without any
modification.
INTERNATIONAL ISO
STANDARD 13885-3
First edition
2020-07
Gel permeation chromatography
(GPC) —
Part 3:
Water as eluent
Chromatographie par perméation de gel (GPC) —
Partie 3: Eluant à l'eau
Reference number
ISO 13885-3:2020(E)
©
ISO 2020
ISO 13885-3:2020(E)
© ISO 2020
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
ISO 13885-3:2020(E)
Contents Page
Foreword .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Apparatus . 2
5.1 Eluent supply . 3
5.2 Pump . 3
5.3 Injection system . 3
5.4 Separation columns . 3
5.5 Column temperature control . 5
5.6 Detector . 6
6 Reagents . 6
7 Calibration of the apparatus . 6
7.1 General . 6
7.2 Specification for the calibration standard . 6
7.3 Preparation of the calibration solutions for injection . 7
7.4 Conditions for calibration runs . 7
7.5 Measurement of elution volume . 7
7.6 Plotting the calibration curve . 7
8 Sampling . 8
9 Preparation for the test . 8
9.1 Preparation of the injection solution . 8
9.2 Preparation of the apparatus . 9
10 Analytical parameters . 9
11 Data acquisition and evaluation . 9
11.1 General . 9
11.2 Calculation of the net chromatogram from the raw data .10
11.2.1 Determination of the baseline .10
11.2.2 Correction of the measured values and of the net chromatogram .10
11.2.3 Evaluation limits .10
11.3 Calculation of the average values .10
11.4 Calculation of the distribution curves .11
12 Precision .12
12.1 General .12
12.2 Repeatability .12
12.3 Reproducibility .12
13 Test report .13
13.1 General .13
13.2 General data on the equipment and settings .13
13.2.1 Data on the equipment used .13
13.2.2 Calibration .13
13.2.3 Evaluation .14
13.3 Special data on the sample .14
Annex A (informative) Conversion of experimental parameters for variant column sizes .15
Annex B (informative) Example of a data sheet for a polymer standard .16
Annex C (informative) Explanations .18
ISO 13885-3:2020(E)
Bibliography .23
iv © ISO 2020 – All rights reserved
ISO 13885-3:2020(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 35, Paints and varnishes.
A list of all parts in the ISO 13885 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
INTERNATIONAL STANDARD ISO 13885-3:2020(E)
Gel permeation chromatography (GPC) —
Part 3:
Water as eluent
WARNING — This document can involve hazardous materials, operations or equipment. It
does not purport to address all of the safety problems, if any, associated with its use. It is the
responsibility of the user to establish appropriate safety and health practices and to ensure
compliance with any national regulatory conditions.
1 Scope
This document specifies the determination of the molar-mass distribution and the average molar
mass values M (number average) and M (weight average) of polymers that are soluble in water by gel
n w
permeation chromatography (GPC).
NOTE Also known as size exclusion chromatography (SEC).
This method is applicable to neutral polymers and polyanions (e.g. polycarboxylates,
polysaccharides, fully hydrolyzed polyvinyl alcohols and high-molecular polyethylene oxides).
It is not applicable to polycations [e.g. polyvinylpyrrolidone, polyvinylpyridine, salts of
poly(diallyl-N,N-dimethyl-azacyclopentane), chitosan].
Despite good solubility in the mobile phase and even though the chromatograms obtained show good
repeatability, it is possible that this method cannot be used with certain polymer types because of
specific interactions (e.g. adsorption) within the sample/eluent/column system (see also Clause 12).
The conditions specified in this document are not applicable to the GPC analysis of polymer samples
with M values greater than 10 g/mol and/or polymers with elution limits outside the calibration
w
range (see 7.6 and Annex C).
This document includes no correction methods (e.g. for the elimination of peak broadening). If absolute
molar mass values are required, an absolute method (e.g. membrane osmometry for M or light
n
scattering for M ) can be used.
w
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 1513, Paints and varnishes — Examination and preparation of test samples
ISO 4618, Paints and varnishes — Terms and definitions
ISO 15528, Paints, varnishes and raw materials for paints and varnishes — Sampling
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 4618 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
ISO 13885-3:2020(E)
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
gel permeation chromatography
GPC
separation of molecules, mainly based on exclusion effects such as differences in size and/or shape of
molecules (size exclusion chromatography) or in charge (ion exclusion chromatography)
[SOURCE: ISO 13885-1:2020, 3.1]
3.2
system peak
signal peculiar to the gel permeation chromatography (3.1) using a refractive index detector
Note 1 to entry: These signals appear at the total penetration limit of the columns and are not part of the sample,
but of the overall system.
[SOURCE: ISO 13885-1:2020, 3.2]
3.3
polycation
water-soluble polymer that carries permanent positive charges or forms temporary cationic structures
due to its pK value under the measurement conditions
B
3.4
polyanion
water-soluble polymer that carries permanent negative charges or forms temporary anionic structures
due to its pK value under the measurement conditions
S
3.5
neutral polymer
water-soluble polymer that carries no charges and forms o-charged groups or structures under the
measurement conditions
4 Principle
The dissolved (molecularly disperse) molecules of a polymer sample are fractionated on a porous
column material, with separation taking place according to the size of the molecule (or, more precisely,
the polymer coil size which forms in this eluent). Small molecules diffuse into the pores of the column
material more frequently and are therefore retarded more than large molecules. Thus, large molecules
are eluted earlier, small molecules later. Under the test conditions given, the elution volume is solely a
function of the coil size of the molecule.
The polymer content of a sample is determined, the sample is then diluted with eluent to give a
concentration of less than 5 g/l and an aliquot of the diluted sample is injected into the GPC system. The
concentration of the molecules eluted from the column is measured in order of decreasing coil size with
a concentration-sensitive detector (typically a differential refractometer). With the aid of a calibration
curve that has been determined for the particular GPC system, the relative molar-mass distribution, the
relative quantities M and M and the heterogeneity or polydispersity M /M are calculated from the
n w w n
chromatogram obtained.
5 Apparatus
The apparatus shall consist of the components shown in Figure 1, which are described below.
All components which come into contact with the eluent or the sample solution shall not exhibit any
adsorption and memory effects and shall not exhibit any expansion effects at the increased temperature.
The eluent described in Clause 6 can cause corrosion in the case of long-term use. For this reason, it is
necessary that high-quality steel, titanium, polyetherketones or polytetrafluoroethylene are used for
2 © ISO 2020 – All rights reserved
ISO 13885-3:2020(E)
all components and that the individual modules are connected to one another by means of capillary
tubes made of high-quality steel or titanium.
Figure 1 — Block diagram of a GPC apparatus
5.1 Eluent supply
The eluent reservoir shall adequately protect the eluent against external influences such as the
atmosphere, if necessary by means of a blanket of inert gas above the liquid level.
The eluent reservoir shall contain a sufficient quantity of the eluent to bring the apparatus to
equilibrium and to carry out several repeat analyses.
The eluent shall be degassed, either before it is introduced into the reservoir or by use of a device fitted
between the reservoir and the pump, to prevent malfunctions of the pump or the formation of bubbles
in the detector. The method of degassing used (e.g. bubble trap, online purging with helium or vacuum
degassing) is open to choice.
5.2 Pump
The pump shall ensure that the eluent flow through the separation column is as smooth and pulse-free
as possible. The flow rate shall be 1 ml/min (see Annex A). To fulfil these requirements, the pump shall
operate at optimum efficiency at this flow rate.
The flow rate of the pump used shall have a variation of max. 0,1 %.
It shall be ensured that the specifications required in 5.6 for detector noise can be adhered to by means
of an appropriate pump structure or a downstream pulsation damper.
5.3 Injection system
The injection system serves to introduce a given amount of the sample solution into the eluent stream
in a rapid and smooth fashion. This introduction can be carried out either manually or automatically.
If the introduction is carried out manually, ensure that the sample loop is filled completely with solvent
before loading with the sample.
Memory effects from the previous sample solution in the injection system shall be avoided by design
measures or by adequate flushing.
5.4 Separation columns
The apparatus shall have one or more columns connected in series and packed with spherical porous
material, the diameter of the pores corresponding to the size of the polymer molecules being analysed.
The packing material typically consists of an acrylate copolymer, produced by a special polymerization
process, with OH functionality and without basic groups. The packing material shall swell only slightly
in the eluent and therefore cannot deform under the pressure developed at the set flow rate.
ISO 13885-3:2020(E)
To keep adsorptive interactions between the packing material surface and the polymer molecules
to be investigated as low as possible, silica phases and modified silica phases shall not be used.
Furthermore, the sample being analysed shall not be changed, either chemically or structurally, within
the chromatographic system.
Certain polymers interact with the surface of the packing material (e.g. by adsorption), and other
effects can sometimes interfere with the GPC separation mechanism. Details of such effects and notes
on possible remedies are discussed in Annex C. If it is intended to compare analyses of such polymers by
different laboratories, the laboratories shall agree on details of the test conditions that are not covered
by this document.
For good repeatability of test results, it is necessary to adhere to the minimum requirements specified
below with regards to peak broadening (expressed in terms of a number of theoretical plates) and
separation efficiency.
a) Number of theoretical plates
The number of theoretical plates, N, shall be determined, for the apparatus used per metre
of column used, from the peak width at half height (see Figure 2). Inject up to 20 µg of ethylene
glycol on to each column under the same test conditions as are used for analysing polymers; the
injection volume shall not exceed 20 µl (see Annex A). Evaluate the chromatogram obtained using
the Formula (1):
V
e
N =×55, 4 × (1)
W L
1/2
where
V is the elution volume measured at the peak maximum;
e
W is the peak width at half height (see Figure 2); the same units shall be used for V and W ;
1/2 e 1/2
L is the length of the column (column combination), in centimetres.
Express as the result the number of theoretical plates per metre of column length. To conform to the
requirements of this document, a column combination shall have at least 10 000 theoretical plates
per metre.
NOTE See Annex C for tailing and fronting (asymmetry) of the peak used to calculate the plate count.
4 © ISO 2020 – All rights reserved
ISO 13885-3:2020(E)
Key
X elution volume
Y peak intensity
1 injection
V elution volume measured at the peak maximum
e
W peak width at half height of the peak
1/2
h maximum peak height
σ standard deviation
Figure 2 — Determination of the number of theoretical plates by the half-height method
b) Separation efficiency
To ensure adequate resolution, the log M versus elution volume, V , calibration curve for the
10 e
column combination used shall not exceed a specified gradient. For the purposes of this document,
the relation given in Formula (2) shall apply in the area of the peak maximum for the polymer
sample under investigation:
VV−
e,M
e,()10×M
x
x
>60, (2)
A
c
where
V is the elution volume for pullulan of molar mass, M , in cubic centimetres;
e,M x
x
V is the elution volume for 10 times that molar mass, in cubic centimetres;
e,1()0×M
x
A is the cross-sectional area of the column, in square centimetres;
c
M is the molar mass of pullulan; it shall be selected such that the peak maximum for the
x
polymer sample under investigation lies approximately halfway between these two
elution volumes.
5.5 Column temperature control
Carry out the test at room temperature (15 °C to 35 °C) or at a higher temperature of max. 40 °C. The
temperature of the column shall not change by more than 1 °C during the analysis (see Annex C).
ISO 13885-3:2020(E)
5.6 Detector
Use a differential refractometer detector. The cell volume shall not exceed 0,010 ml (see Annex A).
NOTE For the restriction to a single detector type, see Annex C.
The detector response obtained using the injection amounts specified in this document shall, at the
lowest setting for electronic damping, exhibit a noise level of less than 1 % of the maximum height of
the polymer peak.
The signals from the detector are recorded by means of an electronic data system (see Clause 11 for
details).
6 Reagents
A solution of sodium chloride (analytical quality), c(NaCl) = 0,1 mol/l, in water of grade 1 according to
ISO 3696 (conductivity: <0,01 mS/m, extinction at 254 nm and 1 cm optical path length: 0,001) is used
as the eluent. The pH value is adjusted to 7,5 to 8,5 using an aqueous sodium hydroxide solution. Other
components shall not be added.
Discard the eluent used to condition the columns or to perform the analyses, and do not return it to the
eluent reservoir.
7 Calibration of the apparatus
7.1 General
The method is not an absolute one and requires calibration with commercially available unbranched
pullulan standards that have been characterized by independent absolute methods. The results for
samples of polymers with different chemical structures are therefore only comparable within groups of
samples of the same type.
Calibrate the GPC apparatus with a series of unbranched pullulan samples of narrow molar mass
distribution (see Annex C) and whose molar masses have been determined by independent, absolute
methods. Glucose, maltose and maltotriose are used for calibration in the low-molar-mass range. The
result is a calibration curve for the evaluation of GPC analyses of unbranched polymethyl methacrylate
samples. If this calibration curve is used to analyse samples of other compositions, containing molecules
with other structures, the results shall be expressed as the “pullulan molar mass equivalents”.
7.2 Specification for the calibration standard
The molar-mass distribution of the standards shall be narrower than the limits given below as a
function of the molar mass at the peak maximum, M :
p
M < 2 000 g/mol M /M ≤ 1,3
p w n
2 000 g/mol ≤ M < 400 000 g/mol M /M ≤ 1,2
p w n
400 000 g/mol ≤ M M /M ≤ 1,5
p w n
The following minimum requirements shall be fulfilled in the characterization of each individual
pullulan standard used for calibration:
a) at least one average molar mass value, M , M or M , shall be determined by an absolute method;
n w z
b) at least one method shall be used to determine the molar-mass distribution;
c) all the parameters involved in the method used shall be indicated;
6 © ISO 2020 – All rights reserved
ISO 13885-3:2020(E)
d) the results and data for each batch analyzed shall be presented in a comprehensible form for the user.
NOTE An example of a data sheet is given in Annex B.
If the calibration standards give a shoulder on either side of the peak, pre-peaks or a tailing peak, the
area represented by these anomalies shall be less than 2,0 % of the peak area, otherwise the calibration
standard shall be rejected.
7.3 Preparation of the calibration solutions for injection
Dissolve the calibration standards in the eluent at ambient temperature without shaking or stirring.
The minimum dissolving time is 12 h. Then homogenize the solutions carefully. Store the solutions at
ambient temperature.
The soaking periods are relatively long during dissolving of the calibration standards. The solutions
shall not be shaken during the soaking phase so that the polymer chains do not tear.
Filter the solutions manually through a 0,2 µm to 0,45 µm membrane filter. If the filter shows signs of
blocking, the solution is unsuitable for calibration purposes.
The solutions shall be used within 48 h.
Several calibration standards may be injected and analyzed at the same time, as long as all the peaks
are separated down to the baseline.
The concentration of the individual calibration standards in the injection solution, as a function of the
molar mass measured at the peak maximum, M , shall be
p
M < 400 000 g/mol 1,0 g/l
p
400 000 g/mol ≤ M 0,5 g/l
p
The quantities injected on to the column shall be matched to the capacity of the column by adjusting the
injection volume and not the concentration. The injection volumes determined in accordance with the
requirements of Clause 10 shall be used both in calibration runs and in sample analyses.
7.4 Conditions for calibration runs
The conditions for a calibration run, with the exception of the concentration of the injection solutions,
shall be identical to those for the sample analyses.
7.5 Measurement of elution volume
The elution volume, V , shall be measured from the start of injection to the point on the baseline at
e
which the peak reaches its maximum height. In determining this point, a baseline drift of 5 % of the
peak height, measured from injection to after the system peaks, is acceptable. If the drift is greater or
the baseline is unsteady in the area of the peak, the analysis shall be repeated.
The elution volume can be measured and checked against an internal standard or an undisturbed
system peak and, if necessary, a linear correction can be made.
NOTE Sulphur or a system peak can be used as an internal standard.
7.6 Plotting the calibration curve
The calibration curve shall be plotted with log M as the ordinate and the elution volume, V , as the
10 p e
abscissa. At least two calibration points shall be measured per decade of molar mass and there shall be
at least five calibration points altogether. In the low-molar-mass range, the calibration curve shall be
extrapolated to the system peaks by using glucose, maltose and maltotriose.
ISO 13885-3:2020(E)
In the high-molar-mass range, the peak of the first calibration standard eluted shall lie before the
high-molar-mass limit of the sample to be analysed, and the position of the exclusion limit shall be
determined.
The results of the calibration runs can be fed into a computer or recorded in the form of a table or in the
form of one or more regression curves. They shall be available at all times in the form of hard copy for
direct checking. Since the evaluation of the chromatograms involves their conversion into differential
distribution curves in which the reciprocal of the first derivative of the calibration curve is required
(see 11.4), the functional relationship log M = f (V or t ) shall be differentiable.
10 e R
To check how well the calibration curve thus produced fits the measurements, the percentage deviation
for each calibration point, given by
MM−
pc,,alibrationvaluep calculated
×100
M
pc, alibrationvalue
shall be plotted against V or t . From this graph, it is assessed whether the positive or negative
e R
deviations are random along the V or t axis. Calibration curve fits which exhibit trends in the deviation
e R
plot over particular elution ranges are unsuitable. If such distributions of residuals cannot be improved
using the regression models (see Annex C) available in a laboratory, the results shall be expected to
contain greater errors and this shall be stated in the test report.
The test for the distribution of residuals need not be carried out on calibration curves obtained by
methods in which the measured points and those of the calibration curve automatically coincide, as
is the case with a connected series of straight lines and with uncompensated spline algorithms. With
these methods, other means shall be used to ensure that the calculated calibration curves contain no
physically impossible areas, e.g. relative extreme values.
8 Sampling
Take a representative sample of the product to be tested, as specified in ISO 15528. Examine and
prepare each sample for testing, as specified in ISO 1513.
9 Preparation for the test
9.1 Preparation of the injection solution
Weigh an aliquot of the polymer sample and dissolve in the eluent (see Clause 6) from the reservoir
of the chromatograph in which it is to be analysed. Store the solution at ambient temperature. The
sample solution is adjusted to pH 7,5 to 8,5 using an aqueous NaOH (c = 0,1 mol/l) solution or aqueous
hydrochloric acid (c = 0,1 mol/l).
The concentration of the injection solution is not an independent quantity. It depends on the total
volume of the column used, and the injection volume. See Clause 10 for details.
The use of ultrasound is not permitted because of the risk of degradation. The use of solution
temperatures higher than 80 °C should be avoided. Exceptions shall be justified in the test report.
As a rule, polymer samples shall be weighed almost free of solvent. If the sample contains solvent and if
it is sensitive, the original solution can be used at its original concentration, or it shall be concentrated
carefully under vacuum at ambient temperature before weighing. The polymer content of the original
solution shall be determined separately; the method used shall be stated in the test report. If such
samples give overlapping system and polymer peaks, the evaluation shall be restricted to the unaffected
polymer area and the evaluation limit stated in the test report in terms of molar mass. When several
samples are analyzed and compared, the evaluation limit selected shall be identical in each case.
Remove insoluble foreign matter (e.g. pigments, extenders and high-impact components) from the
injection solution by suitable methods (e.g. ultracentrifugation, filtration or membrane filtration). Even
8 © ISO 2020 – All rights reserved
ISO 13885-3:2020(E)
if the solution appears clear to the eye, filtration through membrane filters with a pore size between
2 µm and 0,2 µm is always recommended. These operations as well as any precautions taken to ensure
that the concentration of the injection solution is maintained shall be recorded in the test report.
If the sample contains insoluble polymer particles (e.g. microgel), the test report shall expressly point
out that the GPC results refer only to the soluble components. The observations made shall be described.
The injection solutions shall be used within 48 h.
9.2 Preparation of the apparatus
The apparatus shall be operated under the conditions given in Clause 10. First, pump eluent through the
entire apparatus until the detector sensitivity required for the analysis falls below the noise level given
in 5.6 and the baseline condition specified in 7.5 can be expected to be maintained. At this point, the
analyses or, if necessary, the control analyses, can be carried out.
10 Analytical parameters
The concentration of the injection solution shall be 0,5 mg/ml to 5,0 mg/ml.
The injection volume shall be matched to the set of columns used and shall be not more than 100 µl per
(300 × 7,8) mm column; a total value of 250 µl shall not be exceeded (see Annex A).
With narrow molar mass distributions and high molar masses, the elution volume is very sensitive to
the quantity of polymer injected. If anomalous peak shapes are observed with a particular sample, the
concentration of the injection solution shall be repeatedly halved until the effective variation in the
calculated M value has been reduced to below 5 %.
w
Several injections shall be made for each sample. The number of injections made shall be stated in
the test report. The three last injections shall be evaluated individually and the results presented
individually. Their position in the sequence of injections shall be evident.
When comparing analyses carried out by different laboratories, injections shall be made from at least
two solutions that have been prepared separately.
In addition to the usual GPC evaluation of the chromatograms as described in Clause 11, the peak
areas shall be determined and, if necessary, shall be scaled to the same injection amount. The areas of
consecutive analyses shall not differ by more than ±3 % for identical evaluation limits. If a continuous
increase or decrease in the peak areas of consecutive analyses is observed, further analyses shall be
performed until a constant level is reached within the framework of these tolerances. Occurrences of
this kind and other observations that indicate adsorptive interactions between the injected sample and
the surface of the column packing material as described in 5.4 shall be noted in the test report.
NOTE The reason for adding, in comparison to the determination with THF described in ISO 13885-1, this
condition is described in more detail in Annex C.
11 Data acquisition and evaluation
11.1 General
The chromatogram shall be recorded by means of an electronic data acquisition system. Data shall be
stored starting at a point before the exclusion limit for the column system being used and continuing
until the curve returns to the baseline after elution of the last system peak.
The number of the measured points, which shall be equidistant, shall be at least 20 per molar mass decade
of the calibration curve used, and a peak that is to be evaluated shall include at least 25 such points.
The dynamic range of the detector signal between the smallest detectable value and the highest peak in
the chromatogram after subtraction of the baseline shall be at least 1:500.
ISO 13885-3:2020(E)
The raw data obtained from the sample and calibration analyses shall be stored for at least one year to
permit re-evaluation, if necessary.
11.2 Calculation of the net chromatogram from the raw data
11.2.1 Determination of the baseline
The zero signal of the detector (baseline) shall be taken as a straight line between the zone preceding
the exclusion limit and that following the last system peak, i.e. zones in which no elution will take place
in an ideal GPC separation.
The baseline shall coincide with the detector signal in these zones for at least 10 % of the total analysis
time, otherwise the results shall be discarded. If deviations from the baseline thus determined can
be seen in this interval, the results shall also be rejected. The calculations themselves can be made at
points along the baseline that lie within this range on the baseline thus determined.
The plot of the difference between the i-th original measured point and the interpolated baseline point
at the same time between the low- and high-molar-mass cut-offs is referred to in the following as the
net chromatogram.
11.2.2 Correction of the measured values and of the net chromatogram
An adjustment or correction of the or
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